Even with the recent rapid progress in medical technology and treatment cancer is still regarded as one of the most lethal diseases in the world. Further, the global trend of aging society simply speeds up the annual increase in the number of cancer patients. In general, anticancer agents are extremely toxic and they cannot selectively remove cancer cells. Therefore, there has been a long-felt need for the development of an anticancer agent which is very effective but with less toxicity.
Since the development of an automated DNA synthesis in 1980s, research in medical field has been conducted more actively to develop therapeutic agents using siRNA and antisenses which target intracellular mRNAs, aptamers, CpG and decoy which target ribozymes and proteins, etc. [Gleave et al., Nat Rev Cancer. 2005 468-479; Castanotto et al., Nature 2008 426-433; Sullenger et al., Nature 2002 252-258; Kaur et al., Expert Opin Investig Drugs. 2008 43-60; Jurk M et al., BioDrugs. 2007 387-401; Tomita et al., Clin Exp Nephrol. 2007 7-17].
Guanosine-rich oligonucleotides have been known to have an inhibitory activity against growth of broad spectrum of cancer cells. When cancer cells are treated with guanosine-rich oligonucleotides, they bind to particular proteins in the cells, for example, eEF1A, JNK, Ki-ras, Nucleolin, stat3, telomerase, topoisomerase, which are closely associated with cell growth and death, and regulate the cell cycle. These proteins are known to be overexpressed in cancer cells than in normal cells [Christopher R. Ireson et al., Molecular cancer therapy 2006 2957-2962; Naijie Jing et al., Cancer Research 2004 6603-6609; Christophe Marchand et al., The journal of Biological Chemistry 2002 8906-8911].
These guanosine-rich oligonucleotides have a special structure including a triple hydrogen bond with cytosine. They can have a structure with a quadruple strand via an intramolecular or intermolecular binding. Instead of forming a double helix structure by a hydrogen bond between adenosine-thymidine and guanosine-cytidine, four guanosine molecules are located on a single plane to form a hydrogen bond in Hoogsten type thereby forming G-quadruplex. This G-quadruplex is repeated at least once and form a tetrad helical structure.
In general, oligonucleotides have not been favored in developing new drugs due to the low blood stability and cell permeability. However, oligonucleotides with G-quadruplex structure are known to have stably structure and relatively high blood stability and cell permeability [Julian Leon Huppert, Chemical Society Reviews 2008, 37, 1375-1384; Paula J. Bates et al., Experimental and Molecular Pathology (2009) 151-164; Christopher R. Ireson et al., Molecular cancer therapy 2006 2957-2962]. U.S. Pat. No. 7,312,082 teaches that the stability of G-quadruplex depends on the monovalent cations, interchelating agents, and concentration of oligonucleotides or the like. (Haiyan Qi et al., Cancer Res 2006 11808-11816, Anna Arola et al., Current Topics in Medicinal Chemistry 2008 1405-1415).
U.S. Pat. No. 7,314,926 and U.S. Pat. Appl. Publ. No. 2007-105805 disclose that G-Quadruplexes bind to certain proteins, which are expressed on the surface of cancer cells, enter cancer cells by endocytosis, and bind to proteins involved in cell apoptosis thereby inhibiting growth of cancer cells. The cell apoptosis is known to be induced by cytostatic effect rather than cytotoxic effect [Paula J. Bates et al. The journal of Biological Chemistry 1999 26369-26377; Bruna et al., FEBS 2006 1350-1361].
In addition to their inhibitory effect against cancer cell growth, other effects of G-Quadruplex forming oligonucleotides, have been also known. For example, U.S. Pat. No. 5,567,604 discloses antiviral effect; U.S. Pat. No. 6,994,959 discloses effect in immune regulation; and U.S. Pat. Appl. Publ. No. 2007-105805 discloses its role in the treatment of Huntington's disease thus suggesting that they are associated with various biological functions and regulations in the body [Cheryl A. Stoddart et al. Antimicrobial Agents and Chemotherapy 1998 2113-2115; Michael Skogen et al. BMC Neuroscience, 2006 7:65]. Many lines of research have been focused on using G-quadruplex forming oligonucleotides the treatment of various diseases and recently a clinical study has been carried out to prove its potential as an anticancer agent [Paula J. Bates et al. Experimental and Molecular Pathology (2009) 151-164].
AS-1411, a clinical drug developed as a result, is a G-Quadruplex forming oligonucleotide, which binds to nucleolin which are overly expressed in cancer cells thereby exert excellent inhibitory activity against cancer cell growth. Besides, it can considerably reduce its influence on normal cells in the body while increasing its cell apoptotic activity against cancer cells are thus expected to be a new potential anticancer drug [Christopher R. Ireson et al., Mol Cancer Ther. 2006 December; 5(12): 2957-62]. G-quadruplex forming oligonucleotides induce cell apoptosis by inhibiting cell growth. However, there are disadvantages with the G-quadruplex forming oligonucleotides that it is essential to provide a patient with a daily ringer injection for a period of 4-7 days due to their relatively low cytotoxicity, and also there is a burden for the need of combinatorial administration of a chemotherapeutic agent which is highly toxic [Paula J. Bates Et, al. Experimental and Molecular Pathology (2009) 151-164; Christopher R. Ireson et al., Molecular cancer therapy 2006 2957-2962].
To solve the above problems, the inventors of the present invention have made efforts to improve the cell apoptotic effect of the G-quadruplex forming oligonucleotides by introducing therapeutically effective modified nucleic acid having apoptotic effect to the G-quadruplex forming oligonucleotides.
A representative example of therapeutically effective modified nucleic acids is 5-fluorouracil (5-FU). 5-FU was first developed in late 1950.s as an anti-metabolic anticancer agent. It is known to exert anticancer activity by blocking thymidylate synthase [Piedo et al., J Clin Oncol 1988, 1953-1664]. In addition, prodrugs in the form of a 5-fluoropyrimidine nucleoside such as 5-fluorodeoxyuridine(5-FdU), 5-fluorodeoxycytidine(5-FdC), 5-fluorouridine have been used for, the treatment of colorectal cancer, breast cancer, and head and neck cancer for more than 4 decades and they are under clinical experiment [Thomas et al., Clin Exp Pharmacol Physiol. 1998 887-895; Heidelberger et al. Nature 1957 179:663-666; Longley et al., Nat Rev Cancer 2003 330-338; Beumer et al., Cancer Chemother Pharmacol 2008 363-368; Song et al., Clinical cancer research. 1997, 901-909]. Since the synthesis of phosphoramidite preparations, comprising these 5-fluorodeoxyuridine, 5-fluorodeoxycytidine, 5-fluorouridine nucleoside in the oligonucleotides, was enabled, it was made possible to synthesize in a solid phase DNA synthesizer the oligonucleotides containing therapeutic nucleosides [Gmeiner et al., J. Org. Chem. 1994, 5779-5783; Schmidt et al., Nucleic Acids Research, 1992, 2421-2426; Stolarski et al., Biochemistry 1992, 31, 7027-7042].
These therapeutic nucleosides-containing oligonucleotides release 5-FdU and 5-FdC during the enzymatic degaration by exonucleases, and become associated with various kinds of enzymes to be converted into 5-FdUMP as a fully activated form thereby inducing cell apoptosis. Thus obtained oligonucleotides are known to be more cytotoxic than 5-FdU at equal concentration, and are also therapeutically more effective in treating drug-resistant cancer cells [Gmeiner et al., Nucl. Nuct. 1995 243-253].
U.S. Pat. No. 5,457,187 discloses the cytotoxicity of homo poly-FdU oligonucleotide containing 5-fluoro uracil, and U.S. Pat. No. 5,614,505 discloses the cytotoxicity of oligonucleotides comprising FdU. However, these 5-fluoro uracil-containing oligonucleotides do not form G-quadruplex and have a very low blood stability and cell permeability, thus not being suitable to be developed as a drug with respect to their structure and pharmaceutical efficacies.